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Detection of Brucella melitensis and Brucella abortus by DNA amplification
Abstract
Suitable reaction conditions and oligonucleotide primers were sought for the detection of Brucella melitensis and Brucella abortus by the polymerase chain reaction. Primers were chosen from within the coding sequence of a gene encoding a 31 kDa B. abortus antigen. The test was shown to be sensitive, and specificity was demonstrated using DNA derived from a panel of Gram-negative pathogens. There was no detectable difference between B. melitensis and B. abortus in the sensitivity of the reaction or in the size of the amplification product. The technique should be applicable in the diagnosis of brucellosis.
... The search of Brucella spp. DNA in the blood and urine samples was performed by amplification of the gene bspc31 (BAILY et al., 1992). The PCR reaction was performed with a mix containing, 1x of buffer I0 , 3 mM of MgCl 2 , 0.2 mM of dNTP, 1 µM of each primer, 2.5 units of Taq DNA polymerase (Phoneutria Biotecnologia e Serviços LTDA, Brazil) and the DNA template (3 µL) (RICHTZENHAIN et al., 2002). ...
... These results could be due to the low sensitivity of the serological tests used for diagnosis of brucellosis in dogs (MIRANDA et al., 2005;MOL et al., 2020), confirming the need to use more than one diagnostic technique. Furthermore, these results could also be explained by: i) the intermittent bacteremia and shedding in the urine of B. canis in dogs (SERIKAWA et al., 1981;HENSEL et al., 2018); ii) diagnostic in different points of infection, as antibodies are induced only around 21 to 30 days after infection (KAUFFMAN & PETERSEN, 2019); (iii) or even due to infection by other Brucella species (non-rough ones), since the used PCR was not speciesspecific, identifying the presence of DNA of the genus Brucella spp. in the samples (BAILY et al., 1992). Therefore, the use of more than one diagnostic method for the diagnosis of canine brucellosis is preferable to avoid false-negative test results (increasing the sensitivity) and thereby the maintenance of infected dogs in contact with other animals and humans (KEID et al., 2009;MOL et al., 2020). ...
The aim of this study was to assess the prevalence and incidence of canine leptospirosis and brucellosis in Parque Francisco de Assis, a shelter in Lavras, Minas Gerais State, Brazil, as well as the risk factors potentially associated with both diseases. Samples of blood, urine, and sera from all animals were collected in 2019 (n = 329) and 2020 (n = 325 dogs). DNA of Leptospira spp. (urine) and Brucella spp. (urine and blood) were searched by PCR, whereas microagglutination test (MAT) and agar gel immunodiffusion assay (AGID) were performed to identify antibodies anti-Leptospira spp. and anti-rough Brucella spp., respectively. The results showed no positive dogs in PCR for Leptospira spp.; however, a seroincidence of 9.24% was found considering MAT results, with Canicola and Autumnalis being the most common serogroups. The incidence of Brucella spp. PCR-positive animals in the 6 months was 5.62% in the urine and 11.23% in the blood samples, while AGID showed a seroincidence of 11.74% in the period. Overall, our results demonstrated the circulation of Leptospira spp. and Brucella spp. among the dogs from Parque Francisco de Assis, Lavras, Minas Gerais, Brazil, being the weight increase (1.10, 95%CI 1.00-1.21) and neutropenia (3.29, 95%CI 1.60-6.77), the risk factors associated with the occurrence of leptospirosis and brucellosis, respectively. Therefore, brucellosis was identified in the dogs of Parque Francisco de Assis, and the presence of antibodies against Leptospira spp. suggesting contact of the dogs with the pathogen, which represent a risk for the other animals and to the humans in close contact with the positive dogs.
... El objetivo de la presente investigación fue implementar en el LNRB del IPK un ensayo de PCR a punto final que permita la amplificación de un fragmento de 223 pb del gen que codifica para la proteína de membrana externa bcsp de 31Kb, presente en todas las especies y biovares del género Brucella (10). ...
... El límite de detección que se obtiene en esta investigación equivale al genoma de dos células de brucelas, número de microorganismos que se estima que cómo mínimo estarían presentes en 1 mL de sangre periférica de pacientes o animales con brucelosis (10). Sin embargo, investigadores en Grecia reportan que al utilizar los cebadores B4B5 obtienen valores superiores a los de este estudio. ...
Brucellosis is a zoonosis that may cause mild symptoms or severe manifestations. Molecular techniques are very sensitive and specific in the timely and early detection of Brucella spp. DNA, both at the beginning of the infection and in relapses and therapeutic failures. The aim of the present research was to implement a conventional PCR-bcsp that would allow the timely diagnosis of human brucellosis in Cuba, and thus contribute to avoid the occurrence of complications that can lead to disabling sequelae in the patient. A service and system research was carried out at the National Reference Laboratory for Leptospira and Brucella at IPK to evaluate the PCR-bcsp. The parameters of analytical sensitivity and specificity, as well as diagnostic sensitivity and specificity were determined. Conventional PCR was applied to 86 sera from suspected cases of human brucellosis that were negative to the IgM and IgG ELISAs for Brucella. PCR-bcsp presented a detection limit of 6.403 fg/µL. Analytical specificity was 100 %, while the diagnostic sensitivity and specificity showed values of 96.3 % and 100 %, respectively. After its application to sera from serologically negative probable cases of human brucellosis, 85 % positivity was reached. The present research strengthened the microbiological diagnosis of human brucellosis since a molecular tool guaranteeing the early diagnosis of the disease in Cuban environment has been implemented.
... 26 To confirm the presence of Brucella genome in milk samples, real-time PCR was carried out using the BruSpp dtec-qPCR kit (Genetic Analysis Strategies, Spain) followed by conventional PCR based on three Brucella-specific primer pairs as previously described. [27][28][29] Moreover, species-specific primers (Bruceladder) were employed, as previously described. 21 MLVA genotyping of B. melitensis was carried out using the BRUCELLA MLVA-16 Typing Kit (Genoscreen, France), where 16 markers are amplified from purified DNA using four quadruplex PCR reactions; the refined set of 16 VNTR was previously outlined. ...
Background Brucellosis is the most common zoonotic disease in Oman. Studies about genetic diversity of Brucella are limited in the country. This study aimed to genotype Brucella melitensis in human isolates and milk samples using multi-locus variable number tandem repeats analysis (MLVA-14) in Oman. Methods MLVA-14 was employed for forty-nine B. melitensis recovered from human isolates (n = 21), one goat isolate, and milk samples (n = 27). Results Clustering analysis separated the 49 B. melitensis strains into two main clusters including 31 genotypes. In Dhofar Governorate, shared genotypes among different animal species were identified; the same genotypes were found also in human isolates. Moreover, there was a close genetic relationship between human and milk sample strains from Dhofar and AD Dakhiliya Governorates. Phylogeography investigated by Minimum Spanning Tree analysis showed that Omani strains belonged to the East Mediterranean lineage and formed a distinct branch with a close relationship with two strains from the United Arab Emirates. Moreover, eight Omani strains genotyped from milk shared the same MLVA profile as strains from Spain, Portugal, China, India, and Turkey. The caprine isolate was an outlier correlated with a big cluster mostly formed by isolates from China with other strains from Portugal, Kazakhstan, Turkey, Mongolia, Marocco, France and Spain. Conclusions This study highlights the zoonotic nature of B. melitensis transmission from infected livestock to humans and also its circulation among different animal species. The One Health approach is the way to develop policies and programs for disease surveillance and control.
... An 800 bp fragment of 16S rRNA was amplified with primers and cycling conditions as reported by Bricker et al. [9]. Simultaneously PCR targeting 223 bp portion of the 31kDa Brucella cell surface salt extractable protein (bcsp31) gene of Brucella was amplified with primers and cycling conditions as reported by Baily et al. [10]. ...
... applications are the IS711 insertion element and the bcsp31 gene, which codes for a 31-kDa immunogenic outer membrane protein that is conserved across all Brucella species [6]. The most reliable method of diagnosing human brucellosis is still pathogen isolation from blood culture, although ELISA and agglutination-based serological testing are also commonly used. ...
Objective: This study sought to determine the seroprevalence of human brucellosis in patients presenting with acute undifferentiated illness at a tertiary care hospital. Methods: The cross-sectional investigation was done on patients presenting to the outpatient or inpatient Department of Medicine at Government Medical College, Amritsar during the study period, regardless of age group, with an acute undifferentiated febrile illness. A 5 mL of whole blood was extracted in a plain vacutainer from suspected patients and enzyme-linked immunosorbent assay (ELISA) was performed. Results: Out of 100 samples,11 came out positive by ELISA. Four females and seven males tested positive. Three (27.27%) of the 11 samples had immunoglobulin (Ig)G anti-bodies, whereas 8 (72.72%) samples had IgM anti-bodies. Conclusion: Brucellosis is a serious zoonotic illness with consequences for public health. Efforts should be focused on creating and executing efficient animal vaccination programs, as well as on better diagnostic techniques.
... Molecular assays, including a genus-specific bcsp31 PCR assay [39] and a speciesspecific assay, AMOS multiplex PCR [40], were conducted on all swab and milk samples collected in May, as well as on all Brucella isolates identified on both visits. The Bruceladder PCR [41] was performed exclusively on culture-identified Brucella isolates. ...
Brucellosis is a neglected zoonotic disease affecting livestock and humans that remains endemic in Ethiopia. Despite its prevalence, only a few studies have identified Brucella species circulating in livestock in the country. This study aimed to determine the Brucella species responsible for infections in livestock in the Afar region of Ethiopia and characterize the isolates using whole-genome single nucleotide polymorphism (wgSNP) analysis and in silico multi-locus sequence typing (MLST). Comparisons were made between Ethiopian Brucella and regional and global isolates to determine their phylogenetic relationships. Surveys conducted in May and October–November 2022 in six villages of the Amibara district involved the collection of vaginal swabs (n = 231) and milk samples (n = 17) from 32 sheep and 199 goats kept by 143 pastoral households reporting recent abortions in the animals. Brucella melitensis was detected in three sheep and 32 goats, i.e., 15% (35/231) of animals across 20% (29/143) of households using bacterial culture and PCR-based methods (bcsp31, AMOS, and Bruce-ladder multiplex PCR). Of the 35 positive animals, B. melitensis was isolated from 24 swabs, while the remaining 11 were culture-negative and detected only by PCR. The genomic DNA of the 24 isolates was sequenced using Illumina Novaseq 6000 and assembled using the SPAdes pipeline. Nine- and 21-locus MLST identified 23 isolates as genotype ST12, while one isolate could not be typed. The wgSNP-based phylogenetic analysis revealed that the Ethiopian isolates clustered within the African clade and were closely related to isolates from Somalia. Several virulence factors responsible for adhesion, intracellular survival, and regulatory functions were detected in all isolates. No antimicrobial resistance genes associated with resistance to drugs commonly used for treating brucellosis were detected. Since B. melitensis is prevalent in sheep and goats, vaccination with the B. melitensis Rev-1 vaccine is the recommended strategy in these pastoral systems to protect animal and human health.
... 30 Numerous researches were published on the detection of Brucella by PCR, both from pure culture and from field samples mostly of cattle origin. [41][42][43][44] Diagnostic techniques are important for control and extinction of brucellosis; isolation provides the specific diagnosis and it is considered to be the gold standard method. 45 The demerits of the culture technique is that it is tedious, hazardous, and lacks sensitivity and specificity. ...
... Biomolecular analyses were performed to detect cetacean morbillivirus (CeMV) [54], Brucella [55,56], Photobacterium damselae subsp. damselae [57,58], and Toxoplasma gondii [59]. ...
Herpesvirus (HV) is widely distributed among cetacean populations, with the highest prevalence reported in the Mediterranean Sea. In this study, a comprehensive analysis was conducted, including epidemiological, phylogenetic, and pathological aspects, with particular emphasis on neuropathology, to better understand the impact of HV in these animals. Our results show a higher presence of HV in males compared to females, with males exhibiting a greater number of positive tissues. Additionally, adults were more frequently affected by HV infection than juveniles, with no infections detected in calves or neonates. The affected species were striped (Stenella coeruleoalba) and bottlenose dolphins (Tursiops truncatus). The highest positivity rates were observed in the genital system, cerebrum, and skin tissues. Phylogenetic analysis indicated a higher occurrence of Gammaherpesvirus (GHV) sequences but increased genetic diversity within Alphaherpesvirus (AHV). Key neuropathological features included astro-microgliosis (n = 4) and meningitis with minimal to mild perivascular cuffing (n = 2). The presence of concurrent infections with other pathogens, particularly cetacean morbillivirus (CeMV), underscores the complex nature of infectious diseases in cetaceans. However, the presence of lesions at the Central Nervous System (CNS) with molecular positivity for GHV, excluding the involvement of other potential neurotropic agents, would confirm the potential of this HV subfamily to induce neurological damage. Pathological examination identified lesions in other organs that could potentially be associated with HV, characterized by lymphoid depletion and tissue inflammation. These findings enhance our understanding of HV in odontocetes and highlight the need for ongoing research into the factors driving these infections and their broader implications.
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